We propose to develop a high-throughput sequencing methodology for genotyping the Killer Immunoglobulin-like Receptor (KIR) genes using matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry. KIR is 1 of several families of receptors expressed on natural killer (NK) cells, components of the innate immune system specialized for early defense against infection as well as tumors. KIR interact with class I Human Leukocyte Antigen (HLA) ligands which are down regulated in infected and transformed cells. The activation of these early responders of the host immune system is ultimately the result of signal integration from numerous cell surface receptors, including both activating and inhibitory KIR. KIR is diverse in terms of the number, the type, and the combination of genes present in individuals, and display extensive polymorphism at the nucleotide level. These highly variable host genetic factors may significantly affect both the strength and breadth of the immune response to invading viruses. Preliminary data from our lab and others 2-5 support the hypothesis that KIR and their Human Leukocyte Antigen (HLA) ligands play an important role in disease outcome for a number of virally transmitted diseases. We propose to develop an innovative system for complete sequencing of KIR genes from ~30 ng of genomic DMA using the MassCLEAVE(tm) system from Sequenom, Inc. Base-specific cleavage reaction data from amplified target areas are compared to in silica cleavage patterns generated from reference sequences in order to reconstruct the target sequence. Polymorphisms are identified when sequence change leads to changes in cleavage patterns and the resultant characteristics of the mass spectra. A high-throughput and sequence-based method of KIR genotyping would significantly further our understanding of the diversity of this gene complex, and would also contribute significantly to future analysis of KIR in the large cohorts needed for statistical power in disease association studies. [unreadable] [unreadable] [unreadable]